Ectodomain dimerization of proICA512/IA-2 in the endoplasmic reticulum coordinates the conversion of the mature insulin secretory granule form

Introduction: The receptor protein tyrosine phosphatase ICA512/IA-2/RPTPN is an integral component of the insulin secretory granules (SGs) and a major autoantigen in type-1 diabetes. Insulin SG stores are reduced to 50% in mice lacking ICA512. Calpain-mediated cleavage of its cytosolic domain upon SG exocytosis has been proposed as a signaling mechanism for adjusting insulin production to the size and consumption of SG stores. The function of the ICA512 extracellular region, instead, is largely unknown. Resolution of its mature ectodomain (ME ICA512) has revealed that this portion of the extracellular region, similar to mucins, contains a SEA domain and that the recombinant protein can assemble in several dimeric modes in vitro.

Methods: To test if and which among the proposed beta2- and beta4-sheet association interfaces is required for ME ICA512 dimerization in insulinoma cells, ICA512 constructs mutated at critical residues for in vitro dimerization were expressed in INS-1 cells and for ICA512 ectodomain association and glycosylation, investigated by immunoprecipitation and glycosidase enzyme assays.

Results: We confirm that ME ICA512 form dimers, which assemble in the endoplasmic reticulum (ER). We show that both N506 and N524 within ME ICA512 are N-glycosylated, though lack of these modifications neither affected dimerization of proICA512 nor its O-glycosylation, targeting to SGs and conversion. Similar findings were observed upon S508A replacement in the beta2-sheet. Conversely, G553D replacement in the beta4-sheet prevented dimerization and O-glycosylation of proICA512, which accumulated in the ER and underwent extensive proteolysis.

Conclusion: Accordingly, we propose that beta4-sheet mediated dimerization of ME ICA512 is critical for proper folding and exit of ICA512 from the ER, and thus be relevant for SG biogenesis.